Chromatin Immunoprecipitation-quantitative Polymerase Chain Reaction Research Articles

Dysregulation of HDAC9 Represses Trophoblast Cell Migration and Invasion Through TIMP3 Activation in Preeclampsia.

Preeclampsia (PE) is a common disease during pregnancy. It is generally accepted that PE is closely associated with shallow placenta implantation caused by the dysfunction of trophoblast cells. Trophoblasts have been recognized to share histological and behavioral characteristics with cancer cells, and many lines of evidence have emphasized that histone deacetylases (HDACs) are therapeutic targets for cancer treatment with the most promising. However, the roles of HDACs have not been well established in PE. The purpose of this study is investigating the expression of HDACs in preeclamptic placentas and to explore its roles in PE progression. Both mRNA and protein levels of HDAC9 were determined by q-RT-PCR and western blot in normal and preeclamptic placentas. The localization of HDAC9 was performed by immunohistochemistry. Trophoblast cell mobility and proliferation were determined by transwell and MTS assays, respectively. The histone acetylation levels of the tissue inhibitor of metalloproteinases 3 (TIMP3) promoter were detected by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assay. HDAC9 was downregulated in preeclamptic placentas compared with that in normal controls, and it was mainly localized in the nucleus of syncytiotrophoblast cells. HDAC9 knockdown in HTR-8/SVneo cells inhibited cell migration and invasion. The transcriptional level of TIMP3 was upregulated in HDAC9-knockdown HTR-8/SVneo cells because of promoter histone hyperacetylation. Importantly, HDAC9 downregulation can rescue the defects caused by HDAC9 knockdown. HDAC9 promotes trophoblast cell migration and invasion by repressing TIMP3 through promoter histone hypoacetylation. Thus, the findings of our study suggest that dysregulated HDAC9 and TIMP3 are relevant to PE.

Histone methyltransferase NSD2 regulates apoptosis and chemosensitivity in osteosarcoma

Osteosarcoma (OS) is a primary malignant bone tumour. However, the genetic basis for the pathogenesis of OS remains elusive. In this study, we uncovered the role of the histone methyltransferase NSD2 in regulating tumourigenesis and chemosensitivity in OS. We show that NSD2 knockdown leads to increased apoptosis in OS cells in vitro and in vivo. Additionally, NSD2 knockdown significantly enhances the efficacy of cisplatin against OS cells and accordingly inhibits properties associated with cancer stem cells (CSCs). Furthermore, RNA sequencing (RNAseq) and Gene Ontology (GO) analysis revealed that NSD2 promotes transcription of genes associated with negative regulation of apoptotic signalling pathways and CSC properties. The results of chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) assays indicated that NSD2 knockdown leads to decreased H3K36me2 modification at BCL2 and SOX2 loci, thus inhibiting the transcription of these two genes that are closely correlated with apoptosis, CSC properties and chemosensitivity in OS cells. Pathway analysis demonstrated that the ERK and AKT pathways mediate the regulation of OS progression and chemosensitivity by NSD2. Overall, our study is the first to uncover the function of NSD2 in OS chemosensitivity. NSD2 regulates the expression of the apoptosis regulatory proteins BCL2 and SOX2 through the ERK and AKT pathways. Our results suggest that NSD2 is a new target for combined chemotherapy and is a prognostic factor for OS.

Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR).

Chromatin immunoprecipitation (ChIP) is widely used to measure protein-DNA interactions. This protocol outlines a ChIP method used to identify the association of a protein or protein modification (such as a specific histone modification-methylation, acetylation, etc.) of interest with a specific DNA sequence in a target gene in fetal mouse brains on gestational day (GD) 17. Briefly, DNA and proteins are cross-linked (via formaldehyde), and chromatin is sonicated into fragments between 200 and 1000 base pair (bp) long, with an average length of 500bp. The DNA-protein complexes are captured using antibodies directed toward the protein or protein modification of interest. These immunoprecipitated complexes are retrieved using agarose beads. The DNA-protein cross-links are reversed (via heat and via presence of high salt concentrations), and the ChIP DNA is purified and measured via a quantitative polymerase chain (qPCR) reaction. The results show the association of histone modifications at unknown sites of specific genes of interest, indicating which epigenetic modifications of specific genes may be responsible for the outcome of interest.

Contributions of Nrf2 to Puerarin Prevention of Cardiac Hypertrophy and its Metabolic Enzymes Expression in Rats.

Previous evidence has suggested that puerarin may attenuate cardiac hypertrophy; however, the potential mechanisms have not been determined. Moreover, the use of puerarin is limited by severe adverse events, including intravascular hemolysis. This study used a rat model of abdominal aortic constriction (AAC)-induced cardiac hypertrophy to evaluate the potential mechanisms underlying the attenuating efficacy of puerarin on cardiac hypertrophy, as well as the metabolic mechanisms of puerarin involved. We confirmed that puerarin (50 mg/kg per day) significantly attenuated cardiac hypertrophy, upregulated Nrf2, and decreased Keap1 in the myocardium. Moreover, puerarin significantly promoted Nrf2 nuclear accumulation in parallel with the upregulated downstream proteins, including heme oxygenase 1, glutathione transferase P1, and NAD(P)H:quinone oxidoreductase 1. Similar results were obtained in neonatal rat cardiomyocytes (NRCMs) treated with angiotensin II (Ang II; 1 μM) and puerarin (100 μM), whereas the silencing of Nrf2 abolished the antihypertrophic effects of puerarin. The mRNA and protein levels of UGT1A1 and UGT1A9, enzymes for puerarin metabolism, were significantly increased in the liver and heart tissues of AAC rats and Ang II-treated NRCMs. Interestingly, the silencing of Nrf2 attenuated the puerarin-induced upregulation of UGT1A1 and UGT1A9. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction indicated that the binding of Nrf2 to the promoter region of Ugt1a1 or Ugt1a9 was significantly enhanced in puerarin-treated cardiomyocytes. These results suggest that Nrf2 is the key regulator of antihypertrophic effects and upregulation of the metabolic enzymes UGT1A1 and UGT1A9 of puerarin. The autoregulatory circuits between puerarin and Nrf2-induced UGT1A1/1A9 are beneficial to attenuate adverse effects and maintain the pharmacologic effects of puerarin.

RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA.

BackgroundAlthough transcription is the initial process of gene expression, posttranscriptional gene expression regulation has also played a critical role for fine‐tuning gene expression in a fast, precise, and cost‐effective manner. Although the regulation of sodium channel α‐subunit (SCN5A) mRNA expression has been studied at both transcriptional and pre‐mRNA splicing levels, the molecular mechanisms governing SCN5A mRNA expression are far from clear.Methods and ResultsHerein, we show that, as evidenced by ribonucleoprotein immunoprecipitation assay, RNA binding protein Hu antigen R/ELAV like RNA binding protein 1 (HuR/ELAVL1) and myocyte enhancer factor‐2C (MEF2C) transcription factor mRNA are associated. HuR positively regulated transcription factor MEF2C mRNA expression by protecting its mRNA from degradation. As demonstrated by both chromatin immunoprecipitation–quantitative polymerase chain reaction assay and an electrophoretic mobility shift assay, MEF2C enhanced SCN5A transcription by binding to a putative MEF2C binding site within SCN5A promoter region. Overexpression of HuR increased the expression of SCN5A mRNA, and this effect was attenuated by the presence of MEF2C small interfering RNA in cardiomyocytes.ConclusionsIn conclusion, our results suggested that HuR participates in a combined network at the DNA and RNA levels that regulates SCN5A mRNA expression. HuR upregulates MEF2C mRNA expression by protecting MEF2C mRNA from degradation, and consequently, the elevated MEF2C enhances SCN5A mRNA transcription.

Redox-Sensitive Transcription Factor NRF2 Enhances Trophoblast Differentiation via Induction of miR-1246 and Aromatase.

Dysregulation of human trophoblast invasion and differentiation with placental hypoxia can result in preeclampsia, a hypertensive disorder of pregnancy. Herein, we characterized the role and regulation of miR-1246, which is markedly induced during human syncytiotrophoblast differentiation. miR-1246 targets GSK3β and AXIN2, inhibitors of WNT/β-catenin signaling, which is crucial for placental development, and is predicted to target JARID2, which promotes silencing of developmentally regulated genes. Human cytotrophoblasts cultured in 20% O2 spontaneously differentiate to syncytiotrophoblast with induction of hCYP191A/aromatase, a marker of differentiation. miR-1246 was induced >150-fold during syncytiotrophoblast differentiation in 20% O2, whereas targets-GSK3β, AXIN2, and JARID2-were significantly decreased. However, when cytotrophoblasts were cultured in 2% O2, miR-1246 and aromatase induction were prevented. miR-1246 was significantly decreased in placentas of women with severe preeclampsia, whereas AXIN2, GSK3β, and JARID2 were increased, compared with normotensive subjects. To identify factors that regulate miR-1246, we investigated the redox-regulated transcription factor NRF2, which has predicted binding sites in the miR-1246 promoter. Intriguingly, NRF2 messenger RNA was upregulated during syncytiotrophoblast differentiation and significantly reduced by hypoxia and in preeclamptic placentas. Moreover, NRF2 knockdown in cytotrophoblasts inhibited induction of miR-1246 and hCYP19A1, as well as transcription factors C/EBPβ and PPARγ, which are implicated in placental differentiation. Using chromatin immunoprecipitation-quantitative polymerase chain reaction, we found that binding of endogenous NRF2 to the miR-1246 and hCYP191A promoters increased during syncytiotrophoblast differentiation. Thus, NRF2 promotes syncytiotrophoblast differentiation by inducing C/EBPβ, PPARγ, hCYP19A1, and miR-1246, which targets WNT inhibitors and JARID2 and is dysregulated in preeclampsia.

P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts

Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes. HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl4 to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology. Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4;HSC activation and collagen deposition were reduced inlivers of p300F/Fcre mice compared with control mice. In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.

Long Noncoding RNA Meg3 Regulates Mafa Expression in Mouse Beta Cells by Inactivating Rad21, Smc3 or Sin3α

Background/Aims: The main pathogenic mechanism of diabetes is a decrease in the number of islet beta cells or a decline in their function. Recent studies have shown that pancreatic long noncoding RNAs (lncRNAs) have a high degree of tissue specificity and may be involved in the maintenance of islet cells function and the development of diabetes. The aim of this study was to investigate the molecular regulatory mechanism of mouse maternal expressed gene 3 (Meg3) in insulin biosynthesis in pancreatic islets. Methods: Chromatin immunoprecipitation–quantitative polymerase chain reaction (qPCR) and RNA immunoprecipitation–qPCR were used to investigate the molecular mechanism of lncRNA Meg3 in insulin biosynthesis by regulating v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker in the MIN6 beta cell line. Further, the expression levels of Meg3, Ezh2, MafA, Rad21, Smc3, and Sin3α were analyzed in vivo and in vitro by RT-PCR and western blotting. Results: Intranuclear lncRNA Meg3 can bind EZH2, a methyltransferase belonging to the Polycomb repressive complex-2, in pancreatic islet cells. In addition, knockdown of Ezh2 can also inhibit the expression of MafA and Ins2, while expression levels of Rad21, Smc3, and Sin3α are upregulated, by interfering with Ezh2 or Meg3 in pancreatic beta cells. Knockdown of Meg3 resulted in the loss of EZH2 binding and H3K27 trimethylation occupancy of Rad21, Smc3, and Sin3α promoter regions. The inhibition of Rad21, Smc3, or Sin3α, which directly act on the MafA promoter, leads to upregulated expression of MafA in both MIN6 cells and mouse islets. Moreover, the synthesis and secretion of insulin were increased by inhibition of these transcription factors. Conclusions: Pancreatic lncRNA Meg3 can epigenetically regulate the expression of Rad21, Smc3, and Sin3α via EZH2-driven H3K27 methylation. By inhibiting the expression of Rad21, Smc3, or Sin3α, Meg3 promotes the expression of MafA and affects the production of insulin.

The Wnt/β-catenin and PI3K/Akt signaling pathways promote EMT in gastric cancer by epigenetic regulation via H3 lysine 27 acetylation.

In this study, we investigated the underlying mechanism of the phosphoinositide 3-kinase/Akt- and Wnt/β-catenin-mediated promotion of epithelial-to-mesenchymal transition by epigenetic regulation of histone acetylation in gastric cancer. First, we used immunohistochemistry to detect the expression of phosphorylated Akt, phosphorylated glycogen synthase kinase 3 beta, and β-catenin in gastric cancer tissues and adjacent tissues. In addition, we confirmed that the phosphoinositide 3-kinase/Akt and Wnt/β-catenin signaling pathways were correlated with tumorigenesis, progression, and maintenance of gastric cancer using the phosphoinositide 3-kinase inhibitor LY294002 and an inhibitor of the β-catenin/TCF4 complex, FH535. Epithelial-to-mesenchymal transition-related gene expression was measured by western blotting and quantitative real-time polymerase chain reaction assays. Furthermore, we detected the acetylation of histone H3 lysine 4 and lysine 27 using the FH535 and LY294002 inhibitors at different concentrations for 24 and 48 h. Finally, chromatin immunoprecipitation-quantitative polymerase chain reaction was performed to detect the specific binding of H3K27ac to the promoter of the epithelial-to-mesenchymal transition-related factor, Twist. Taken together, abnormal activation of the phosphoinositide 3-kinase/Akt and Wnt/β-catenin signaling pathway was correlated with the gastric cancer progression and contributed to epithelial-to-mesenchymal transition regulation by controlling histone acetylation.

A Role for Progesterone-Regulated sFRP4 Expression in Uterine Leiomyomas.

Context:Despite progesterone’s key role in uterine smooth muscle tumorigenesis, the mechanisms by which it promotes the growth of uterine leiomyomas remain poorly understood.Objective:The aim of this study was to identify gene products mediating the effects of progesterone in uterine leiomyomas.Design:Gene expression profiling was used to identify putative progesterone-regulated genes differentially expressed in uterine leiomyomas, which were then studied in vitro.Methods:Gene expression was comprehensively profiled with the Illumina WG BeadChip (version 2.6) and analyzed with a bioinformatic algorithm that integrates known protein–protein interactions. Genomic binding sites for progesterone receptor (PR) were interrogated by chromatin immunoprecipitation—quantitative polymerase chain reaction (ChIP-qPCR). Small interfering RNA was used to study gene function in primary cell lines.Results:Our analyses identified secreted Frizzled-related protein 4 (sFRP4) as a key gene product functionally linked to PR activation whose expression was 2.6 times higher in leiomyomas than myometrium (n = 26, P < 0.01) and 2.5 times higher during the proliferative phase of the menstrual cycle (n = 26, P < 0.01). Direct binding between PR and sFRP4 promoter was observed by ChIP-qPCR. Robust overexpression of sFRP4 was also observed in primary cultures derived from leiomyoma. Progesterone preferentially inhibited sFRP4 expression and secretion in leiomyoma cultures in a dose-dependent manner sensitized by estradiol. Knockdown of sFRP4 inhibited proliferation and apoptosis in primary cultures of both myometrium and leiomyoma.Conclusions:Overexpression of sFRP4 is a robust, progesterone-regulated feature of leiomyomas that increases smooth muscle proliferation. More work is needed to elucidate how progesterone’s ability to modulate sFRP4 expression contributes to uterine smooth muscle tumorigenesis.

Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1.

Previous studies from this group demonstrated that insulin gene enhancer binding protein ISL-1 (Islet-1) specifically induces the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte-like cells through histone acetylation. However, the underlying mechanisms remain unclear. In the present study, the role of the histone acetylation and DNA methylation on the regulatory mechanism of the Islet-1 was further investigated by methylation-specific polymerase chain reaction (PCR), chromatin immunoprecipitation quantitative PCR and western blot analysis. The results demonstrated that Islet-1 upregulated expression of general control of amino acid biosynthesis protein 5 (Gcn5) and enhanced the binding of Gcn5 to the promoters of GATA binding protein 4 (GATA4) and NK2 homeobox 5 (Nkx2.5). In addition, Islet-1 downregulated DNA methyltransferase (DNMT)-1 expression and reduced its binding to the GATA4 promoter. In contrast, the amount of DNMT-1 binding on Nkx2.5 did not match the expression trend. Therefore, it was concluded that Islet-1 may influence the histone acetylation and DNA methylation of GATA4 promoter region via Gcn5 and DNMT-1 during the MSC differentiation into cardiomyocyte-like cells, thus prompting the expression of GATA4. The Nkx2.5 was likely only affected by histone acetylation instead of DNA methylation. The present study demonstrated that Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through a specific interaction between histone acetylation and DNA methylation on regulating GATA4.

The NOTCH Ligand JAG1 Regulates GDNF Expression in Sertoli Cells.

In the seminiferous epithelium of the testis, Sertoli cells are key niche cells directing proliferation and differentiation of spermatogonial stem cells (SSCs) into spermatozoa. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), which is essential for SSC self-renewal and progenitor expansion. While the role of GDNF in the testis stem cell niche is established, little is known about how this factor is regulated. Our previous studies on NOTCH activity in Sertoli cells demonstrated a role of this pathway in limiting stem/progenitor cell numbers, thus ultimately downregulating sperm cell output. In this study we demonstrate through a double-mutant mouse model that NOTCH signaling in Sertoli cells functions solely through the canonical pathway. Further, we demonstrate through Dual luciferase assay and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) analysis that the NOTCH targets HES1 and HEY1, which are transcriptional repressors, directly downregulate GDNF expression by binding to the Gdnf promoter, thus antagonizing the effects of FSH/cAMP. Finally, we demonstrate that testicular stem/progenitors cells are activating NOTCH signaling in Sertoli cells in vivo and in vitro through the NOTCH ligand JAG1 at their surface, indicating that these cells may ensure their own homeostasis through negative feedback regulation.

Lysine demethylase 2A promotes stemness and angiogenesis of breast cancer by upregulating Jagged1.

Alterations of histone methylation dynamically regulated by methyltransferases and demethylases are frequently found in human cancers. Here, we showed that expression of lysine demethylase 2A (KDM2A) is markedly increased in human breast cancer and its overexpression is associated with tumor progression and poor prognosis. Knockdown of KDM2A in breast cancer cells reduced proliferation but not viability. Gene set enrichment analysis revealed that inhibition of KDM2A down-regulates angiogenic genes with concurrent reduction of Jagged1 (JAG1), NOTCH1 and HEY1 in the NOTCH signaling. Chromatin immunoprecipitation- quantitative polymerase chain reaction (ChIP-qPCR) demonstrated the binding of KDM2A to the JAG1 promoter and the increase of methylation of Lys-36 of histone H3 (H3K36) in KDM2A-depleted MDA-MB-231 cells. Tumorsphere formation was significantly reduced in KDM2A-depleted cells which could be reversed by ectopic expression of JAG1. A selective KDM2A inhibitor daminozide also decreased the number of tumorsphere and the number of CD24−/CD44hi cells. In addition, daminozide acted synergistically with cisplatin in cell killing. We identified SOX2 as a direct transcriptional target of KDM2A to promote cancer stemness. Depletion of KDM2A in MDA-MB-231 cells attenuated NOTCH activation and tube formation in co-cultured endothelial cells. Two pro-angiogenic factors JAG1 and PDGFA are key mediators for KDM2A to enhance angiogenesis. Finally, inhibition of KDM2A significantly decreased tumor growth and angiogenesis in orthotopic animal experiments. Collectively, we conclude that KDM2A functions as an oncogene in breast cancer by upregulating JAG1 to promote stemness, chemoresistance and angiogenesis.

De-repression of CSP-1 activates adaptive responses to antifungal azoles.

Antifungal azoles are the major drugs that are used to treat fungal infections. This study found that in response to antifungal azole stress, Neurospora crassa could activate the transcriptional responses of many genes and increase azole resistance by reducing the level of conidial separation 1 (CSP-1), a global transcription repressor, at azole-responsive genes. The expression of csp-1 was directly activated by the transcription factors WC-1 and WC-2. Upon ketoconazole (KTC) stress, the transcript levels of wc-1 and wc-2 were not changed, but csp-1 transcription rapidly declined. A chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed a rapid reduction in the WC-2 enrichment at the csp-1 promoter upon KTC treatment, which might be responsible for the KTC-induced csp-1 downregulation. Deletion of csp-1 increased resistance to KTC and voriconazole, while csp-1 overexpression increased KTC susceptibility. CSP-1 transcriptionally repressed a number of azole-responsive genes, including genes encoding the azole target ERG11, the azole efflux pump CDR4, and the sterol C-22 desaturase ERG5. Deletion of csp-1 also reduced the KTC-induced accumulation of ergosterol intermediates, eburicol, and 14α-methyl-3,6-diol. CSP-1 orthologs are widely present in filamentous fungi, and an Aspergillus fumigatus mutant in which the csp-1 was deleted was resistant to itraconazole.

Characterization of Hydroxymethylation Patterns in the Promoter of β-globin Clusters in Murine Fetal Livers.

DNA methylation of 5-methylcytosine (5mC) is a key epigenetic regulator in mammals; the dynamic balance between methylation and demethylation affects the transcriptional activity of β-globin. However, the dynamic cytosine methylation of β-globin in vivo during the different stages of embryogenesis and in developing liver has not been fully established. 5-Hydroxymethylcytosine (5hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5mC by the ten-eleven translocation (TET) family and it has not been fully identified in β-globin clusters. Here, we determined the 5hmC modifications in the promoter of murine β-globin from fetal livers during normal embryonic development with the methods of bisulfite (BS) and oxidative bisulfite (oxBS)-based pyrosequencing techniques, with the combination of methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR). The results characterized the 5hmC modification at the CpG sites of -426, -388, and -151 of ɛ(y) promoters and -50 and -487 CpG of β(h1) from transcriptional start sites from E15.5 and E17.5 livers, while 5hmC modification was not observed in the adult β-globin promoters. These observations were validated by the induction of TET transcription after being treated with a potent demethylating agent 5-azacytidine, and TET-mediated hydroxymethylation of ɛ(y) and β(h1) from E13.5 livers was also confirmed in our study. These results suggested the 5hmC modification in promoters of ɛ(y) and β(h1) and indicated that the 5hmC modification is essential for the β-globin switching before the embryonic globin reactivation.

Characterization of combinatorial histone modifications on lineage-affiliated genes during hematopoietic stem cell myeloid commitment.

Hematopoietic stem cells (HSCs) are multipotent stem cells capable of self-renewal and multilineage differentiation. Mechanisms regulating the maintenance of HSCs' multipotency and differentiation are still unclear. In this study, we observed the role of combinatorial histone modification pattern in the maintenance of HSCs' pluripotency and differentiation. HSCs (CD34(+)CD38(low)) were collected from human umbilical cord blood and induced to differentiate to committed cells in vitro. The histone modifications on lineage-specific transcription factors/genes in multipotent HSCs and differentiated progenies, including megakaryocytes, granulocytes, and erythrocytes, were analyzed by chromatin immunoprecipitation-quantitative polymerase chain reaction. Our results showed that a certain level of acH4 and acH3 together with high level of H3K4me2, low level of H3K4me3, and a certain level of H3K9me3 and H3K27me3 were present in lineage-specific genes in CD34(+)CD38(low) HSCs. As CD34(+)CD38(low) cells differentiated into granulocytes, erythroid cells, and megakaryocytes, the modification levels of acH3, acH4, and H3K4me2 on lineage-specific genes remained the same or elevated, while H3K4me3 level was increased greatly. At the same time, H3K9me3 and H3K27me3 modification levels became lower. In non-lineage-specific genes, the acH3 and acH4 levels were decreased, and H3K4me3 level remained at low level, while H3K9me3 and H3K27me3 levels were increased drastically. Our data suggest that combinatorial histone modification patterns have implicated function in maintaining the multipotency of HSCs and keeping the accuracy of gene expression program during HSC differentiation.

Lrrc34, a novel nucleolar protein, interacts with npm1 and ncl and has an impact on pluripotent stem cells.

The gene Lrrc34 (leucine rich repeat containing 34) is highly expressed in pluripotent stem cells and its expression is strongly downregulated upon differentiation. These results let us to suggest a role for Lrrc34 in the regulation and maintenance of pluripotency. Expression analyses revealed that Lrrc34 is predominantly expressed in pluripotent stem cells and has an impact on the expression of known pluripotency genes, such as Oct4. Methylation studies of the Lrrc34 promoter showed a hypomethylation in undifferentiated stem cells and chromatin immunoprecipitation-quantitative polymerase chain reaction analyses of histone modifications revealed an enrichment of activating histone modifications on the Lrrc34 promoter region. Further, we could verify the nucleolus-the place of ribosome biogenesis-as the major subcellular localization of the LRRC34 protein. We have verified the interaction of LRRC34 with two major nucleolar proteins, Nucleophosmin and Nucleolin, by two independent methods, suggesting a role for Lrrc34 in ribosome biogenesis of pluripotent stem cells. In conclusion, LRRC34 is a novel nucleolar protein that is predominantly expressed in pluripotent stem cells. Its altered expression has an impact on pluripotency-regulating genes and it interacts with proteins known to be involved in ribosome biogenesis. Therefore we suggest a role for Lrrc34 in ribosome biogenesis of pluripotent stem cells.

Characterization of an archaeal two-component system that regulates methanogenesis in Methanosaeta harundinacea.

Two-component signal transduction systems (TCSs) are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase) and its cognate response regulator. FilR1 carries a receiver (REC) domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP-qPCR) using an anti-FilR1 antibody, FilR1 was shown to form in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of M. harundinacea.

Age-Related Brain Expression and Regulation of the Chemokine CCL4/MIP-1β in APP/PS1 Double-Transgenic Mice

The detrimental effect of activation of the chemokine CCL4/MIP-1β on neuronal integrity in patients with HIV-associated dementia has directed attention to the potential role of CCL4 expression and regulation in Alzheimer disease. Here, we show that CCL4 mRNA and protein are overexpressed in the brains of APPswe/PS1ΔE9 (APP/PS1) double-transgenic mice, a model of cerebral amyloid deposition; expression was minimal in brains from nontransgenic littermates or single-mutant controls. Increased levels of CCL4 mRNA and protein directly correlated with the age-related progression of cerebral amyloid-β (Aβ) levels in APP/PS1 mice. We also found significantly increased expression of activating transcription factor 3 (ATF3), which was positively correlated with age-related Aβ deposition and CCL4 in the brains of APP/PS1 mice. Results from chromatin immunoprecipitation-quantitative polymerase chain reaction confirmed that ATF3 binds to the promoter region of the CCL4 gene, consistent with a potential role in regulating CCL4 transcription. Finally, elevated ATF3 mRNA expression in APP/PS1 brains was associated with hypomethylation of the ATF3 gene promoter region. These observations prompt the testable hypothesis for future study that CCL4 overexpression, regulated in part by hypomethylation of the ATF3 gene, may contribute to neuropathologic progression associated with amyloid deposition in Alzheimer disease.